Biomarker profiling of plasma from acute coronary syndrome patients. Application of ProteinChip Array analysis.

AIM: Acute coronary syndrome (ACS) is one of the leading causes of death in the world and remains a complex pathophysiologic process involving inflammatory, hemostatic and vascular processes. The purpose of this study was to identify unique proteomic biomarkers present in patients with ACS using a newly developed proteomic profiling technique, surface enhanced laser desorption/ionization (SELDI). METHODS: Citrated plasma samples obtained from clinically confirmed cases of ACS (n=100) and age matched controls (n=25) were profiled using SELDI-time of flight (TOF)-mass spectrometry (Ciphergen Biosystems, Freemont, CA, USA). A strong anion exchange (SAX) ProteinChip Array was used to profile these samples. In addition to spectra profiles, protein density plots were be obtained from the generated molecular profile. RESULTS: The SELDI profile in the molecular weight (MW) range of 0-150 kDa revealed a prominent 66.3 kDa albumin peak along with several distinct components at 28 kDa, 13.7 kDa and 6.5 kDa. Additional minor molecular components were also noted in the lower MW range (<6 kDa). There was a cluster of peaks between 10 and 12 kDa that were unique to the patients with ACS; about 1/3 of the ACS patients exhibited these peaks as evident in the ProteinChip Array spectrum. None of the age-matched controls exhibited the peaks in this MW range, nor did the normal human plasma pool that was used as an additional control. The relative intensity of these novel molecular components in the range of 10-12 kDa represent unique proteins/peptides which are generated in specific pathologic states associated with ACS. CONCLUSIONS: These observations suggest that patients with ACS have a unique cluster of molecular components that are present in their SELDI profile. It might be possible to use these patterns to identify high-risk patients who may be more susceptible to the development of unstable plaque, which may eventually lead to myocardial infarction. Identification and characterization of these molecular components will also help in the understanding of the pathogenesis of ACS. These unique peaks may represent pathologic proteins, novel inflammatory mediators or protease cleavage products. Further studies need to be done to better characterize and identify these molecular components and their pathologic role in ACS.

A novel nuclear protein, 5qNCA (LOC51780) is a candidate for the myeloid leukemia tumor suppressor gene on chromosome 5 band q31.

Interstitial deletion or loss of chromosome 5, del(5q) or -5, is a frequent finding in myeloid leukemias and myelodysplasias, suggesting the presence of a tumor suppressor gene within the deleted region. In our search for this gene, we identified a candidate, 5qNCA (LOC51780), which lies within a consistently-deleted segment of 5q31. 5qNCA expresses a 7.2-kb transcript with a 5286-bp open reading frame which is present at high levels in heart, skeletal muscle, kidney, placenta, and liver as well as CD34+ cells and AML cell lines. 5qNCA encodes a 191-kD nuclear protein which contains a highly-conserved C-terminus containing a zinc finger with the unique spacing Cys-X2-Cys-X7-His-X2-Cys-X2-Cys-X4-Cys-X2-Cys and a jmjC domain, which is often found in proteins that regulate chromatin remodeling. Expression of 5qNCA in a del(5q) cell line results in suppression of clonogenic growth. Preliminary sequence results in AML and MDS samples and cell lines has revealed a possible mutation in the KG-1 cell line resulting in a THR to ALA substitution that has not been found in over 100 normal alleles to date. We propose 5qNCA is a good candidate for the del(5q) tumor suppressor gene based on its predicted function and growth suppressive activities, and suggest that further mutational and functional study of this interesting gene is warranted.

Cytogenetic and molecular diagnosis of chromosome 5 deletions in myelodysplasia.

Deletions of chromosome 5, del(5q), are frequently observed in myelodysplasia (MDS). We evaluated molecular detection of loss of heterozygosity (LOH) as a diagnostic method to detect del(5q) in a series of 60 MDS cases at a single institution. LOH was compared to cytogenetics on the same clinical specimen, resolving ambiguous cases by fluorescent in situ hybridization (FISH) and additional LOH. There was poor concordance between molecular and cytogenetic results, but most discrepancies could be resolved by FISH and additional LOH. Molecular analysis was of low sensitivity because most cases contained a relatively high proportion of cells without del(5q), but it was accurate, while cytogenetics overestimated the proportion of cells with del(5q) and failed to detect some cases with complex rearrangements. Minor clones were detected both by FISH and LOH. Overall, we found an incidence of 23% (14 of 60 cases) for del(5q) in MDS. The results also suggest that there is a high degree of genetic heterogeneity in the cellular population of MDS. Although del(5q) is common in MDS, it may not be present in all cells, leading to diagnostic challenges.

Human mortalin (HSPA9): a candidate for the myeloid leukemia tumor suppressor gene on 5q31.

Human mortalin (HSPA9) was originally identified by its close homology to murine mortalins, which play important roles in cellular senescence. The two murine genes, mot-1 and mot-2, differ in only two amino acid residues, but have opposite functions in cellular immortalization. HSPA9 was recently localized to chromosome 5, band q31, a region that is frequently deleted in myeloid leukemias and myelodysplasia (MDS), making it a candidate tumor suppressor gene, which is consistent with the biological function of its murine homologue. To evaluate mortalin in this capacity, its expression in normal and leukemic cell lines was investigated, and its genomic structure was determined in order to facilitate mutation detection. RT-PCR and Northern blot analysis revealed a broad distribution in normal tissues and in leukemia cell lines, producing a single 2.8 kb transcript. Genomic characterization showed that the gene spans 18 kb, and consisted of 17 exons with boundaries that were almost identical to its murine counterpart. Using intron-based primers to flank each exon, sequence of the complete protein-coding regions was obtained for three AML cell lines, including two lines with chromosome 5 loss (KG-1 and HL-60) and one without (AML-193) compared to normal DNA. No mutations were identified although one conservative nucleotide sequence variant was observed in exon 16. We have shown that mortalin is highly conserved in genomic structure as well as sequence, and the designed primers will be suitable for future studies to detect mutations in clinical samples.

Low intelligence but not attention deficit hyperactivity disorder is associated with resistance to thyroid hormone caused by mutation R316H in the thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is a syndrome of reduced responsiveness of tissues to thyroid hormone. The clinical manifestations are variable and 46-50% of children with RTH have attention deficit hyperactivity disorder (ADD). We present a new family with RTH (F120) found to have a mutation R316H in the thyroid hormone receptor beta (TR beta) gene identical for that reported in an unrelated family. Assignment of the mutant allele and haplotyping based on CA repeat polymorphism were done on 16 family members. Semistructured diagnostic interviews and psychometric testing were used to determine the psychiatric diagnosis of 12 family members by examiners blinded to the genotype. Three subjects were identified to have the R316H allele as well as mildly elevated free T4 index (168 +/- 12; normal range 77-135) and nonsuppressed TSH (4.1 +/- 1.7 mU/L). Only 2 of the subjects with RTH were found to have ADD, while one family member homozygous for the wild type TR beta and normal thyroid function tests also had ADD. Unaffected family members had higher full scale intelligence quotients (IQ) (93 +/- 7) than any of the 3 family members with RTH (77 +/- 5, p = 0.006). These data do not support the genetic linkage of ADD and RTH, but do suggest that RTH is associated with lower IQ scores that may confer a high likelihood of exhibiting ADD symptoms.

A new point mutation (C446R) in the thyroid hormone receptor-beta gene of a family with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is a condition of impaired end-organ responsiveness to thyroid hormone characterized by goiter and elevated thyroid hormone levels with an inappropriately normal TSH. RTH has been associated with mutations in the thyroid hormone receptor-beta (TR beta) gene. We report studies carried out in 21 members of a family (F119), 12 of whom exhibited the RTH phenotype. A point mutation was detected in the T3-binding domain of the TR beta gene. It resulted in replacement of the normal cysteine-446 with an arginine (C446R) that has not been previously reported. The clinical characteristics of this family are similar to those reported in other families with RTH, namely goiter, tachycardia, and learning disabilities. Thyroid function tests are also typical of other subjects with RTH. The mean values (+/- SD) in untreated affected subjects compared to those in unaffected family members were: free T4 index, 250 +/- 21 vs. 108 +/- 13; total T3, 4.3 +/- 0.4 vs. 2.4 +/- 0.4 nmol/L; and TSH, 4.5 +/- 1.1 vs. 2.4 +/- 1.1 mU/L. DNA samples from 18 family members were screened for the TR beta mutation, which results in the loss of a BsmI restriction site, and each of the 11 subjects with abnormal thyroid function tests were heterozygous for the mutant allele. The mutant TR beta expressed in Cos-I cells did not bind T3 (Ka of C446R/wild-type, < 0.05). T3 at a concentration up to 100 nmol/L failed to enhance the transactivation of a reporter gene, and the mutant receptor inhibited the T3-mediated transcriptional activation of the wild-type TR beta.

Resistance to thyroid hormone in subjects from two unrelated families is associated with a point mutation in the thyroid hormone receptor beta gene resulting in the replacement of the normal proline 453 with serine.

Resistance to thyroid hormone (RTH) is a condition of impaired tissue responsiveness to thyroid hormone characterized by elevated free thyroid hormone levels in serum accompanied by nonsuppressed TSH. RTH has been associated with mutations in the thyroid hormone receptor (TR) beta gene. We report studies carried out in 9 members of a family (F94) of Jewish ethnic origin and a single subject of Mexican origin. All subjects fulfilling the criteria of RTH (6 of family F94 and one of family F27) had the same point mutation in the T3-binding domain on one of the two alleles of the TR beta gene. This mutation resulted in the replacement of the normal proline-453 with serine (P453S). Nevertheless, the clinical characteristics of affected members of each of the two families differed as did the severity of hormonal resistance in terms of responses to the administration of L-T3. Genetic studies indicate that the same mutation occurred independently in each of the two families.